Method for applying metformin and sodium butyrate in K-ras mutation cancer treatment

ABSTRACT

A method for using metformin and sodium butyrate in combination to treat a cancer patient with K-ras mutation is disclosed. When administering the pharmaceutical composition to a cancer patient, Metformin and sodium butyrate offer cooperatively therapeutic efficacy. The present invention also discloses a pharmaceutical composition and a pharmaceutical kit containing both aforementioned. The application of the method, the pharmaceutical composition and the pharmaceutical kit of the present invention are advantageous for improving the treatment effect to cancer patients with K-ras mutation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application relates to and claims the benefit of the co-pendingU.S. non-provisional application Ser. No. 14/157,873, filed Jan. 17,2014, which claims priority under 35 U.S.C. § 119(a) on PatentApplication No. 102117107 filed in Taiwan, Republic of China on May 14,2013, the contents of these applications are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Field of Invention

This invention relates to a method for treating cancer patients, inparticular to methods for treating cancer patients with K-ras mutation.

Related Art

Metformin is isolated from Galega offivinalis, and Galega officinaliswas used for the treatment of diabetes for centuries. In the 1920s, theguanidine compound was discovered in Galega extracts, and, by animalexperiments, the guanidine compound has been confirmed to be the mainactive ingredient to reduce blood glucose concentration. So far, we'veknown the mechanism of metformin or the medically acceptable saltthereof is to enhance glucose uptake and carbohydrates catabolism inmuscle cells, and to suppress glucose production by the liver. Metforminthus has an antihyperglycemic effect, and has been widely used intreating type 2 (non-insulin dependent) diabetes mellitus.

Some patients with the type 2 diabetes mellitus have extraordinary highrisk of having one of the three cancers at the same time: colorectalcancer, breast cancer and pancreatic cancer. Some studies show thatadministration of metformin or the pharmaceutically acceptable saltthereof can generally reduce the risk of developing cancer in thepatients with type 2 diabetes mellitus. In other words, among thepatients with type 2 diabetes mellitus, those who are administered withmetformin or the pharmaceutically acceptable salt thereof have less riskof developing colorectal cancer, breast cancer or pancreatic cancer thanthose who are not administered.

On the other hand, the sodium butyrate is a histone deacetylaseinhibitor (HDACi). The phenomenon of the histone deacetylase (HDAC)overexpression widely exists in many kinds of cancer; therefore, thesodium butyrate is considered as a substance with a great therapeuticpotential, and applied in many cell or animal experiments. Currentstudies have found that the sodium butyrate inhibits growth of somespecific cancer cell and induces apoptosis of some specific cancer cell;thereby the sodium butyrate has an efficacy of slowing tumor growth.Furthermore, sodium butyrate is also considered as a protective agent ofthe intestinal tract, which can repair the intestinal mucosa and treatthe inflammation of the intestinal tract, thereby to reduce the risk ofcolitis oncogenesis. Furthermore, sodium butyrate also has been found tobe able to prevent the developing of breast cancer.

In summary, currently studies confirm that metformin or thepharmaceutically acceptable salt thereof, or the sodium butyrate isindividually effective for some specific cancer, to a certain degree.However, the therapeutic effect of each compound is only has theirrespective effect. Besides, metformin is still mostly used in thetreatment of diabetes. As for reducing the morbidity of colorectalcancer, breast cancer or pancreatic cancer, due to the mechanism is notclear, such efficacy has not been expected. Therefore, further studiesof metformin and sodium butyrate still make no breakthrough for now.

However, especially for cancer patients with K-ras mutation, currentdrug has no significant therapeutic effect, and the clinical studiesalso confirm using metformin or the pharmaceutically acceptable saltthereof, or using sodium butyrate, is not much help for relieving orameliorating the symptoms of the K-ras mutation cancer.

SUMMARY OF THE INVENTION

Only using metformin or the pharmaceutically acceptable salt thereof, oronly using the sodium butyrate to some specific cancer and tumors hassome efficacy, but still no research reports or literatures point outthe combined use of metformin and sodium butyrate. However, applicantshave found the therapeutic efficacy of combined use of metformin andsodium butyrate is much better than using metformin or sodium butyrateindividually. Hence, the present invent discloses a pharmaceuticalcomposition, kit and method for applying metformin and sodium butyrate,to improve the therapeutic efficacy of the K-ras mutation cancer.

In details, the present invention discloses a method for using ametformin and a sodium butyrate for treating a cancer patient with K-rasmutation, wherein when the pharmaceutical composition administered tothe cancer patient, the metformin and the sodium butyrate offercooperatively therapeutic efficacy. In the present invention, the methodas mentioned also includes a use for using a metformin and a sodiumbutyrate for treating a cancer patient with K-ras mutation.

The present invention also discloses a pharmaceutical composition fortreating a cancer patient with K-ras mutation, the compositioncomprising a metformin and a sodium butyrate, wherein when thepharmaceutical composition administered to the cancer patient, themetformin and the sodium butyrate offer cooperatively therapeuticefficacy.

The present invention also discloses a pharmaceutical kit for treating acancer patient with K-ras mutation, the pharmaceutical kit comprising ametformin along with a first pharmaceutically acceptable carrier,diluent or excipient and a sodium butyrate along with a secondpharmaceutically acceptable carrier, diluent or excipient, wherein whenthe pharmaceutical kit is administered to the cancer patient, theMetformin and the sodium butyrate offer cooperatively therapeuticefficacy.

In order to describe more clearly about the technical features of thepresent invention, the following defines some specific terms, andfurther illustrate the detail of the present invention. In the presentinvention, the metformin and sodium butyrate are used in combination fortreating cancer patients with K-ras mutation, and “use (or used, using). . . in combination” means using these two ingredients both: metforminand sodium butyrate. They can be made into a pharmaceutical composition,which includes both of the metformin and the sodium butyrate. They canalso be made into a pharmaceutical kit (it also be called combined drugor combined medicine), which includes two kinds of medicine, oneingredient along with metformin and another ingredient along with sodiumbutyrate, and the two kinds of medicine are used at the same time.Preferably, the pharmaceutical composition or the pharmaceutical kitindividually has an effective amount of metformin and an effectiveamount of sodium butyrate, wherein the effective amount means the doseof these two ingredients which are combined to use can producesynergistic effects.

The “K-ras mutation” used here means the mutation of a KRAS gene, whichis a kind of oncogene, and the KRAS gene can be classified into thenormal (or wide-type) KRAS gene and the mutant KRAS gene. The normalKRAS gene suppresses tumor growth, but the mutant KRAS gene promotescell proliferation, and causes tumor generation. In addition, morestudies show that K-ras mutation is found at high rates in lung cancer,pancreatic cancer and colon cancer. The generally therapeutic drugs forthe treatment of above cancer patients are limited, thus, by theclinical tests, the cancer patients are divided into two categories: ofnon K-ras mutation and of K-ras mutation.

In addition, the “synergistic effect” herein means when using two ormore substances in combination, it produces an effect greater than thesum of their individual effect (which includes producing the sameefficacy or reducing the adverse reactions). The experiments of presentinvention show that the therapeutic efficacy with combinedadministrating metformin and sodium butyrate to the cancer patients withK-ras mutation is better than only using metformin or sodium butyrateindividually.

The “treat” means it can reduce, relieve, ameliorate, improve, or affectcancer and the symptoms thereof, for example, inhibiting or treatingpain or bleeding induced by cancer, or slow or reverse tumorprogression.

The “metformin” means the metformin itself, and also includes thederivatives thereof which are chemically or biologically modified orsubstituted and still retain the same or similarity properties ofmetformin. Of course, the “metformin” also includes the medicallyacceptable salt of metformin, which is based on the acidic group or thebase of its chemical structure, for example, the metforminhydrochloride.

Further, the compounds or substances which are mentioned andsubstantially related to the present invention, which include anypharmaceutically acceptable form thereof. The pharmaceuticallyacceptable form can be but not limited to the diastereomer, enantiomerand all types of isomers, salt, free form, solvent, prodrug, polymorphand racemic mixture thereof.

In the present invention, when the metformin and the sodium butyrate areused in combination, the metformin is administered preferably at a dosebetween 130 mg and 1,000 mg, and the sodium butyrate is administeredpreferably at a dose between 40 mg and 300 mg, and the weight ratio ofthe metformin to the sodium butyrate is 4:1 (the ratio in molarity is2.67:1). Of course, the above administered dose and weight ratio canalso be the compositing ratio of the ingredients in the pharmaceuticalcomposition, or the compounding ratio of the pharmaceutical kit whenactual used.

In the present invention, the cancer patients with K-ras mutation can benot limited to lung cancer patients, pancreatic cancer patients, rectalcancer patients or colon cancer patients with K-ras mutation, fortreating tumors, delaying tumor progression or prevent tumor generation.

In the present invention, when the metformin and the sodium butyrate areused in combination, both cooperatively modulate the metabotropicglutamate signaling pathway in cancer cells to treat the cancer patient.

As mentioned above, the method, the pharmaceutical composition and thepharmaceutical kit of the present invention, based on using themetformin and sodium butyrate in combination, are advantageous forimproving the treatment effect to cancer patients with K-ras mutation.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thesubsequent detailed description and accompanying drawings, which aregiven by way of illustration only, and thus are not limitative of thepresent invention and wherein:

FIG. 1 is a bar chart illustrating the apoptosis results of the treatedHCT116 cells analyzed by flow cytometer according to experiment 2.

FIG. 2 is a bar chart illustrating the apoptosis results of the treatedHCT116 cells analyzed by flow cytometer according to experiment 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be apparent from the following detaileddescription, which proceeds with reference to the accompanying drawings,wherein the same references relate to the same elements.

The present invention discloses a method for using metformin and sodiumbutyrate for treating a cancer patient with K-ras mutation. In theembodiment, metformin and sodium butyrate can be made into apharmaceutical composition, and then the pharmaceutical composition isadministered to patients. And the synthesis methods of metformin andsodium butyrate are well-understood by the person having ordinary skillin the art, and are not repeated here. The pharmaceutical compositionabove mentioned can be in a solid, liquid or half-liquid dosage form,and the present invention is not limited to this way. When applying insolid dosage form, preferably, the metformin and the sodium butyrate areuniformly mixed to make a tablet, which is advantageous topharmaceutical effect. But in the solid dosage form, the metformin andthe sodium butyrate are not limited to uniformly mixed. In other words,in the same dosage form of the pharmaceutical composition, the metforminand the sodium butyrate can be mixed to a degree rather than uniformly,and even can be unmixed. For example, the pharmaceutical composition canbe made as a tablet or capsule, and one part of it is metformin, and theother part of it is sodium butyrate. Preferably, in other embodiments,the commercial metformin powder and sodium butyrate powder can directlybe bought from the manufactures as raw materials, and the pharmaceuticalcomposition is made as a powder agent by mixing both powder of metforminand sodium butyrate.

Of course, for the convenience of manufacture, packaging oradministration, the pharmaceutical composition preferably may comprise apharmaceutically acceptable carrier, diluent, excipient or combinationthereof, to facilitate the manufacture of the suitable combined dosageform or recipe form. Wherein, the pharmaceutically acceptable carrier,diluent or excipient, for example, can be a known magnesium carbonate,magnesium stearate, talc, sugar, lactose, or combinations thereof.

Generally, the suitable dose range of metformin for treating cancer iswell-understood by the person having ordinary skill in the art, which isalso the applied dose range of metformin when metformin and sodiumbutyrate are used in combination for treatment. For example, therecommended daily dose of metformin is between 0.2 mg and 2,000 mg perkilogram of body weight of the cancer patient with K-ras mutation; whenused in combination, the dose of metformin is preferably controlledbetween130 mg and 1,000 mg, and the dose of sodium butyrate ispreferably controlled between 40 mg and 300 mg. Within the above range,the therapeutic efficacy of various doses might have considerable gapsbetween each other. So that, the preferable dose for treatment can bereferred to the following experiments, in which the weight ratio of themetformin to the sodium butyrate is 4:1.

Of course, the dose of metformin or sodium butyrate may change with theactive ingredient, administrating route or individual needs andphysiological condition. In general, the oral administration needs highdose, but the dose used in the early treatment is relatively low.

Each recipe may contain a dosage unit of the pharmaceutical composition.In other words, a recipe contains sufficient dose to generatetherapeutic efficacy for treating a cancer patient with K-ras mutation,to facilitate direct administering. In the embodiment, each powder agentcontains a dosage unit of the pharmaceutical composition. Of course, inother embodiments, a dosage unit may also be consisted of a plurality ofpharmaceutical compositions which are dispended into many sub-units orsub-packages, For example, it is dispended into two to three tablets orcapsules, which are packaged in the same blister pack.

Furthermore, according to the method of the present invention, when thepharmaceutical composition is administered to the cancer patient,metformin and sodium butyrate produce synergistic effect, to improve orenhance the therapeutic efficacy of individually using metformin orsodium butyrate to treat the cancer patients with K-ras mutation. Then,it reaches to eliminate, inhibit, improve, ameliorate, relieve, orprevent the cancers and the symptoms thereof, or delay, stop, reversetumor progression, or achieve the similar medical effects of the aboveobjects

The present invention also discloses a pharmaceutical composition fortreating a cancer patient with K-ras mutation, the compositioncomprising a metformin and a sodium butyrate. However, thepharmaceutical compositions are substantially the same withpharmaceutical compositions above mentioned, and it can be referred tothe aforementioned, and is not repeated here.

The present invention also discloses a pharmaceutical kit, whichcomprising a metformin along with a first pharmaceutically acceptablecarrier, diluent or excipient and a sodium butyrate along with a secondpharmaceutically acceptable carrier, diluent or excipient. Wherein,metformin, sodium butyrate and other relative descriptions can bereferred as aforementioned, and the insufficient or unexplained partwill be illustrated here.

The first or second pharmaceutically acceptable carrier, diluent,excipient or the combination thereof can be substances or materialsunderstood in the technical field of the present invention, and themanufacturing method to combine metformin and sodium butyrate and thedosage form thereof, are well-understood by the person having ordinaryskill in the art. And in a embodiment, the pharmaceutical kit haveseparate packages or containers, such as blister packs, respectivelyreceiving or storing the tablets made by metformin along with a firstpharmaceutically acceptable excipient and the tablets made by sodiumbutyrate along with a second pharmaceutically acceptable excipient, foroffering patients to take at the same time, or administering to patientsafter modulated. Particularly, the pharmaceutical kit is limited to usemetformin and sodium butyrate in combination. However, the use incombination might be either the metformin or the sodium butyrate isfirst administered to the patients, and before losing the efficacythereof, the other is administered to the patients. The time lapsebetween the two substances are administered can be 1 hour to 3 days. Itis preferable to administer both of the substances at the same time topatients, or administer one of the substances then immediatelyadministering the other.

As mentioned above, according to the method, the pharmaceuticalcomposition and the pharmaceutical kit of the present invention, usingthe metformin and sodium butyrate in combination is advantageous forimproving the treatment effect to cancer patients with K-ras mutation.

Experiment 1: Preparation of Pharmaceutical Composition

In the present invention, metformin was purchased from China Chemical &Pharmaceutical Co., Ltd., Taiwan, and sodium butyrate was purchased fromMerck Ltd., Taiwan. About 331 mg of metformin (2 mM) and about 83 mg of(0.75 mM) of sodium butyrate was weight in ambient temperature, and thenmetformin powder and sodium butyrate powder was mixed in a weight ratioof the metformin to the sodium butyrate that is 4:1 (the ratio inmolarity is 2.67:1), which is a dosage unit. Then, the dosage unit ispackaged as a powder agent or into a cachet, and stored at roomtemperature.

Experiment 2: The Pharmaceutical Composition of Metformin and SodiumButyrate has Synergistic Effect on Cancer Cells' Apoptosis

The human colon cancer cell HCT116 (cell line with K-ras mutation) iscultured in the McCoy's 5A with 10% fetal bovine serum. When the HCT116cells are cultured to the appropriate number, then the adherent HCT116cells are suspended, and 2.5×105 HCT116 cells per well are seeded in a6-wells dish. After incubated for 12 hours, sodium butyrate with a finalconcentration of 0.75 mM, metformin with a final concentration of 2 mM,and the pharmaceutical composition with final concentration of 0.75 mMmetformin and 2 mM sodium butyrate are respectively added to the 6-wellsdish as above mentioned.

In this embodiment, the final concentration as mentioned means theconcentration of respective substances after added in the medium forcell culture, and therefore the medium respectively contains 0.75 mMsodium butyrate, 2 mM metformin, or 0.75 mM sodium butyrate and 2 mMmetformin.

After respectably added the above compounds to HCT116 cells, the 6-welldish is incubated in an incubator of 37° C. for 72 hours. After that,the medium and compounds in each well are sequentially removed, and eachwell is washed by Phosphate Buffered Saline (PBS), then the HCT116 cellsare harvested, by treating trypsin-EDTA, for apoptosis analysis.

The apoptosis analysis is based on the Annexin V apoptosis detection kit(BD Pharmingen) for analysis the exposure of phosphatidylserine, and theprotocol follows the accompanying manual. In brief, the treated HCT116cells are washed three times with PBS, and then immediately stained withAnnexin V/Propidium Iodide (PI). 1% fetal bovine serum albumin (BSA) isfirst added to the treated cells. After subsequently added 222.5 μl ofbinding buffer, 10 μl of PI and 2.5 μl of Annexin V-FITC are directlythen added to stain the cells, followed by immediate incubation for 10minutes at low temperature in the dark. The percentage of the apoptosisis calculated by the flow cytometry and FACSCalibur, the analysissoftware thereof.

FIG. 1 is a bar chart illustrating the apoptosis results of the treatedHCT116 cells according to experiment 2. With reference to FIG. 1, thebar labeled with “control group” represents the apoptotic rate of HCT116cells treated by the same processing steps but without adding metforminand sodium butyrate, and the bar labeled with “SB” or “Metformin”represents the apoptotic rate of HCT116 cells treated by the sameprocessing steps but only added either sodium butyrate (SB) ormetformin, and the bar labeled with “SB and Metformin” means adding bothof sodium butyrate and Metformin. The bar chart shows that the apoptoticrate of HCT116 cells only added either sodium butyrate (SB) or metforminincreases to about 20%, which is improved about 15% compared to thecontrol group. Moreover, when using the sodium butyrate and themetformin in combination, the apoptotic rate of HCT116 cells increasesto 35%, which is significantly improved about 30% compared to thecontrol group. Therefore, according to the experiment 2, it shows thatthe pharmaceutical compositions using metformin and sodium butyrate incombination do have better toxic effect, resulting in increasingapoptotic rate, due to the synergistic effect of using sodium butyrateand metformin in combination.

Experiment 3: The apoptosis in Cancer Cells Treated by PharmaceuticalCompositions with Different Final Concentration of Metformin and SodiumButyrate

The protocol and conditions are almost the same as experiment 2, butdifferent doses (concentrations) of sodium butyrate, metformin, or thepharmaceutical composition of metformin and sodium butyrate are used totreat HCT116 cells. In this experiment, for easy to understanding, thegroup of only using sodium butyrate to treat the HCT116 cells is calledgroup A, and the group of only using metformin to treat the HCT116 cellsis called group B, and the group of using the pharmaceuticalcompositions of metformin and sodium butyrate to treat the HCT116 cellsis called group C as follows, wherein, groups A, B and C are furtherdivided into seven subgroups with different concentrations (in thisexperiment, that means final concentration), as shown in the followingtable:

Number A1 A2 A3 A4 A5 A6 A7 Sodium butyrate 3 1.5 0.75 0.375 0.188 0.0940.047 (mM) Number B1 B2 B3 B4 B5 B6 B7 Metformin (mM) 8 4 2 1 0.5 0.250.125 Number C1 C2 C3 C4 C5 C6 C7 Sodium butyrate 3 1.5 0.75 0.375 0.1880.094 0.047 (mM) Metformin (mM) 8 4 2 1 0.5 0.25 0.125

FIG. 2 is a bar chart illustrating the apoptosis results of the treatedHCT116 cells according to experiment 3. With reference to FIG. 2, thedifferent patterns in the bar charts respectively represent A, B, Cgroup, and the numbers at X-axis represent corresponding groups ofdifferent concentrations of compounds or pharmaceutical compositions (asshown in above tables), and the numbers at Y-axis represent the ratio ofsurviving cells in each group relative to the control group. Thesurviving cells are calculated by the flow cytometer and the analysissoftware thereof, FACSCalibur.

The bar chart shows that using metformin and sodium butyrate incombination can reduce the survival rate of the HCT116 cells, which isK-ras mutation cell line, and it means the cytotoxic effect is stronger(higher proportion of apoptosis), and better than other groups withindividual using metformin or sodium butyrate. Particularly using 0.375mM to 3 mM of sodium butyrate and 1 mM to 8 mM of metformin incombination has much better synergistic effect. The results show thatthe pharmaceutical composition produces the better therapeutic efficacy,due to the synergetic effect of metformin and sodium butyrate.

The weight ratio of the metformin to the sodium butyrate is 4:1 (theratio in molarity is 2.67:1) in the experiments mentioned above, but thepresent invention is not limited to it. In other experiments, the doseof metformin and sodium butyrate used in combination can be 16 mM ofmetformin and each concentration thereof 2-fold serially diluted for 7times, combined with 50 mM of sodium butyrate and each concentrationthereof 2-fold serially diluted for 7 times thereof, and any combinationthereof.

Details are shown in the following table:

Metformin (mM) 16 8 4 2 1 0.5 0.25 Sodium butyrate (mM) 50 25 12.5 6.243.12 1.56 0.78

In other words, the dose of using metformin and sodium butyrate incombination can be any combination of each the concentrations, which areshowed in the above table. For example, 16 mM metformin can respectivelybe combined with 50, 25, 12.5, 6.24, 3.12, 1.56 or 0.78 mM sodiumbutyrate, and other ways of combination are not repeated here. It alsooffers improving or enhancing therapeutic efficacy to the cancer ortumor patients with K-ras mutation, so the weight ratio of the metforminto the sodium butyrate of the present invention is not necessarily 4:1.

Although the invention has been described with reference to specificembodiments, this description is not meant to be construed in a limitingsense. Various modifications of the disclosed embodiments, as well asalternative embodiments, will be apparent to persons skilled in the art.It is, therefore, contemplated that the appended claims will cover allmodifications that fall within the true scope of the invention.

What is claimed is:
 1. A method for treating a cancer patient with K-rasmutation, comprising administering to the subject an effective amount ofmetformin and an effective amount of sodium butyrate in a weight ratioof 4:1.
 2. The method according to claim 1, wherein the cancer patientis a lung cancer patient, a pancreatic cancer patient, a rectal cancerpatient or a colon cancer patient.
 3. The method according to claim 1,wherein the metformin and the sodium butyrate act synergistically intreating the cancer patient.
 4. The method according to claim 1, whereinthe metformin is administered at a dose between 130 mg and 1,000 mg, andthe sodium butyrate is administered at a dose between 40 mg and 300 mg.5. The method according to claim 1, wherein the metformin and the sodiumbutyrate cooperatively modulate the metabotropic glutamate signalingpathway in cancer cells to treat the cancer patient.